Genetically engineered attenuated double-stranded RNA viruses

ABSTRACT

The present invention relates to engineering attenuated viruses by altering a non-coding region or the coding sequence of a viral gene. Alterations of the non-coding regions which regulate transcription and/or replication are described. These alterations result in the down-regulation of the viral gene and an attenuation of the virus, either by the production of defective particles during replication, or by reducing the number of progeny virions produced during viral replication. Alterations of viral coding sequences are also described which result in a recombinant or chimeric attenuated virus.

This is a division of application Ser. No. 08/318,794 filed Dec. 20,1994, now U.S. Pat. No. 6,022,726, which is a continuation-in-part ofapplication Ser. No. 07/868,596, filed Apr. 14, 1992 now abandoned.

This is a Continuation-In-Part of Ser. No. 07/868,596 filed Apr. 14,1992, which is incorporated by reference herein in its entirety.

The work reflected in this application was supported, in part, by agrant from the National Institutes of Health, and the Government mayhave certain rights in the invention.

1. INTRODUCTION

The present invention relates to engineering attenuated viruses byaltering a non-coding region or the coding sequence of a viral gene.Alterations of the non-coding regions which regulate transcriptionand/or replication are described. These alterations result in thedown-regulation of the viral gene and an attenuation of the virus,either by the production of defective particles during replication, orby reducing the number of progeny virions produced during viralreplication. Alterations of viral coding sequences are also describedwhich result in a recombinant or chimeric attenuated virus.

2. BACKGROUND OF THE INVENTION

Inactivated virus vaccines are prepared by “killing” the viral pathogen,e.g., by heat or formalin treatment, so that it is not capable ofreplication. Inactivated vaccines have limited utility because they donot provide long lasting immunity and, therefore, afford limitedprotection. An alternative approach for producing virus vaccinesinvolves the use of attenuated live virus vaccines. Attenuated virusesare capable of replication but are not pathogenic, and, therefore,provide for longer lasting immunity and afford greater protection.However, the conventional methods for producing attenuated virusesinvolve the chance isolation of host range mutants, many of which aretemperature sensitive; e.g., the virus is passaged through unnaturalhosts, and progeny viruses which are immunogenic, yet not pathogenic,are selected.

Recombinant DNA technology and genetic engineering techniques, intheory, would afford a superior approach to producing an attenuatedvirus since specific mutations could be deliberately engineered into theviral genome. However, the genetic alterations required for attenuationof viruses are not known or predictable. In general, the attempts to userecombinant DNA technology to engineer viral vaccines have mostly beendirected to the production of subunit vaccines which contain only theprotein subunits of the pathogen involved in the immune response,expressed in recombinant viral vectors such as vaccinia virus orbaculovirus. More recently, recombinant DNA techniques have beenutilized in an attempt to produce herpes virus deletion mutants orpolioviruses which mimic attenuated viruses found in nature or knownhost range mutants. Until very recently, the negative strand RNA viruseswere not amenable to site-specific manipulation at all, and thus couldnot be genetically engineered.

3. SUMMARY OF THE INVENTION

The present invention relates to the production of attenuated virusesusing recombinant DNA techniques. At least two approaches forengineering attenuated viruses are described. One approach involvesengineering alterations of a non-coding region of the virus thatregulates transcription and/or replication of a viral gene so that atleast one of the viral genes is down regulated. This approach may beapplied to a number of different viruses and is advantageously used toengineer segmented viruses where down regulation of the synthesis of oneviral segment results in the generation of defective particles duringeach round of viral replication so that the progeny viruses demonstrateattenuated characteristics. In non-segmented viruses, the downregulation of a viral gene can result in a decrease in the number ofinfectious virions produced during replication, so that the virusdemonstrates attenuated characteristics.

A second approach involves engineering alterations of a viral codingregion so that the viral protein expressed is altered by the insertion,deletion or substitution of an amino acid residue or an epitope and anattenuated chimeric virus is produced.

The attenuated viruses of the invention may advantageously be usedsafely in live virus vaccine formulation. As used herein, the term“attenuated” virus refers to a virus which is infectious but notpathogenic; or an infectious virus which may or may not be pathogenic,but which either produces defective particles during each round ofreplication or produces fewer progeny virions than does thecorresponding wild type virus during replication. Pathogenic viruseswhich are engineered to produce defective particles or a reduced numberof progeny virions are “attenuated” in that even though the virus iscapable of causing disease, the titers of virus obtained in a vaccinatedindividual will provide only subclinical levels of infection.

4. DESCRIPTION OF THE FIGURES

FIG. 1. The noncoding sequences of the NA segments of influenza A/WSN/33virus and of the NA/B-NS transfectant virus. The 5′- and 3′-terminalsequences are drawn in a panhandle structure, which consists of twobase-paired stems and one mismatched internal-loop in the middle. Thenoncoding nucleotides of the chimeric NA gene of the NA/B-NS virus arederived from the NS gene of influenza B/Lee virus. The large lettersindicate nucleotides in the 5′ and 3′ terminal regions (containing 13and 12 nucleotides, respectively) which are different for the two NAgenes. The panhandle structure of the wild type virus NA gene is dividedinto regions A/D, B/E, and C/F, and that of the attenuated gene intoa/d, b/e, and c/f. Regions B/E and b/e contain the second stem regionsof the NA and the NA/B-NS genes, respectively. The open triangle marksthe altered Kozak sequence in the NA gene of the NA/B-NS virus.

FIGS. 2A-2C. Characterization of the RNA of the NA/B-NS virus. FIG. 2A.RNA electrophoresis. The RNAs extracted from purified viruses wereanalyzed on a 3% polyacrylamide gel containing 7.7 M urea and visualizedby silver staining. Lane 1, RNA of influenza A WSN/33 virus; lane 2, RNAof NA/B-NS transfectant virus; lane 3, RNA obtained by run-offtranscription from plasmid Pt-NA/B-NS which produces the chimeric NARNA. FIG. 2B. Analysis of NA RNA in virions by ribonuclease protectionassay (RPA). 50 ng of RNA extracted from purified virus was used in thehybridization reaction with positive sense NS and NA specific riboprobesas described in Materials and Methods. The protected probes wereelectrophoresed on a 6% acrylamide gel containing 7 M urea. Lane 1:riboprobes without RNase A/T₁ digestion; lane 2: riboprobes followingRNase A/T₁ digestion; lane 3: riboprobes protected by the RNA ofinfluenza A/WSN/33 virus; and lane 4: riboprobes protected by the RNA ofNA/B-NS transfectant virus. FIG. 2C. Quantitation of NA-specific RNA invirus by primer extension. The vRNA extracted from either WSN/33 virus(lane 2) or NA/B-NS transfectant virus (lane 3) was reverse transcribedby Rnase H minus reverse transcriptase using NS and NA segment specificprimers as described in Materials and Methods. The products for the NSRNAs are 195 nt long and those for the NA RNAs approximately 260 ntlong. The products were analyzed on a 6% polyacrylamide gel containing7M urea. Size markers are shown on the left.

FIGS. 3A-3C. Time course of mRNA synthesis in MDBK cells. FIG. 3A.Quantitation of NA- and NS-specific mRNAs at different times p.i. byribonuclease protection assay (RPA). The diagram on the topschematically illustrates the procedure. Both NS and NA probes are minussense, and contain sequences corresponding to the 3′-terminal side ofthe cRNA, flanked by vector sequences at the 3′-terminus as indicated bythe open rectangles. The sizes of the probes are shown on the top, andthe sizes of the resulting products are indicated at the bottom of thediagram. The time points (hrs) are indicated on the top. The positionsof probes and products on the gel are indicated by arrows at the leftand right, respectively. FIG. 3B. Time course of NS-specific MRNAsynthesis. The amount of the NS MRNA was measured by direct counting theradioactivity (counts per minute) in the corresponding band excised fromthe gel shown in FIG. 3A. FIG. 3C. Comparison of NA-specific MRNAsynthesis of transfectant virus and A/WSN/33 virus in infected cells.The amount of mRNA for each time point was determined as described inFIG. 3B.

FIG. 4. Analysis of NA-specific vRNA synthesis in infected cells byprimer extension. RNA isolated from infected cells was reversetranscribed using reverse transcriptase and NA and NS vRNA-specificprimers, as described in Materials and Methods. vRNAs extracted frompurified virus were used as control (right). The resulting products weredisplayed on a 6% acrylamide denaturing gel. The reverse transcripts ofthe NS RNAs are 195 nt long and those of the NA-specific segments areapproximately 260 nt long, as indicated by arrows at the right. Thenumbers on the top indicate the times (hrs) postinfection. Virionrepresents the RNA obtained from purified virus.

FIGS. 5A-5B. Analysis of NA protein in virion and in infected cells.FIG. 5A. Western analysis of NA protein in virion. As described inMaterials and Methods, a monoclonal antibody directed againstcarbohydrate was used to quantitate the glycoprotein in the viruses. Theproteins are indicated by HA0 (uncleaved HA), HA1 (subunit 1 of HA) ANDNA at the right. FIG. 5B. Viral proteins synthesized in infected cells.At different times postinfection (hrs p.i.), the viral proteins werelabelled with ³⁵S[cysteine] for 30 minutes, and analyzed on a 10%Laemmli gel. The different proteins are marked at the right. Theposition of the NA protein is indicated by an arrow. The amount of NAprotein was determined by counting the gel a radioanalytic AMBIS imagingsystem using NP and M1 proteins as controls.

FIG. 6. Vector for construction of chimeric HA molecules. FIG. 6A.Chimeric HA/ME1 malaria construct. FIG. 6B. H1 Chimeric HA/polio H2.

FIG. 7. Schematic Structure of Neuraminidase (NA). Tetrameric NAinserted in viral membrane is depicted. Ct, cytoplasmic tail; TM,transmembrane domain; Stalk, stalk region; Head, the globular domainmost distal to the viral membrane.

FIG. 8. Diagram of neuraminidase mutants. The four domains of theneuraminidase molecule are indicated: CT, cytoplasmic tail; TM,transmembrane domain; Stalk, stalk region and Head, the globular domainmost distal to the viral membrane. The numbering system of the aminoacids is according to Hiti and Nayak, 1982, J. Virol. 41:730-734.Deletions are as indicated, and insertions and mutations in the stalkregion are underlined. The diagram is not drawn to scale. (+) indicatesthat infectious virus was recused following transfection of the mutantRNA.

FIG. 9. Gel analysis of vRNAs extracted from purified viruses. Virusesand vRNAs were prepared as described in Section 9.1. 200 ng virion RNAwas analyzed on a 2.8% polyacrylamide gel containing 7.7 M urea. The RNAsegments were visualized by silver staining as described previously(Enami et al., 1990, Proc. Natl. Acad. Sci. USA 87:3802-3805). Each RNAsegment is indicated at the left. Lane 1, wild type transfected virus.Lane 2, Del 16 mutant virus. Lane 3, Ins 12 mutant virus. Lane 4, Ins 24mutant virus. Lane 5, Ins 41 mutant virus. Arrows indicate the positionof the NA genes in each of the RNA preparations.

FIG. 10. Analysis of vRNA by reverse transcription and PCR. RNAextracted from purified virus was reverse transcribed and the NAgene-specific transcripts were then amplified by PCR as described inSection 9.1. The PCR products labelled with gamma [³²P] ATP wereanalyzed on a 6% polyacrylamide gel containing 7 M urea (Luo et al.,1991, J. Virol. 65:2861-2867). The expected products for wild type NA,Del 18m, Del 23N and Del 28N mutant NAs were 214, 160, 145 and 130nucleotides long, respectively. PhiX174 RF DNA/Hae III fragments (BRL,Bethesda, MD) were labelled with gamma ³²[P] ATP and were used as sizemarkers. The sizes of the DNA fragments are indicated at the left. Lane1, size marker. Lane 2, product of wild type NA (T3NAmod). Lane 3,product of Del 18m mutant NA. Lane 4, product of Del 23N mutant NA. Lane5, product of Del 28N mutant NA. Arrows indicated the position of thePCR products.

FIGS. 11A-11B. Growth curves of transfectant viruses in MDBK (FIG. 11A.)and MDCK. (FIG. 11B.) cells. Cells were infected with wild type ormutant viruses at the m.o.i. of 0.001 and virus titers of supernatantcollected at the indicated times were determined as described in Section9.1.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to genetically engineered attenuatedviruses, and methods for their production. Recombinant DNA techniquescan be utilized to engineer site specific mutations into one or morenoncoding regions of the viral genome which result in thedown-regulation of one or more viral genes. Alternatively, recombinantDNA techniques can be used to engineer a mutation, including but notlimited to an insertion, deletion, or substitution of an amino acidresidue(s) or an epitope(s) into a coding region of the viral genome sothat altered or chimeric viral proteins are expressed by the engineeredvirus. The invention is based, in part, on the discovery that the downregulation of a viral gene in segmented viruses results in theproduction of defective particles at each round of replication, so thatthe virus demonstrates attenuated characteristics. In non-segmentedviruses, the down-regulation of a viral gene may result in theproduction of fewer progeny virions than would be generated by thecorresponding wild type virus. The alterations of the viral proteinsdescribed also result in attenuation for reasons which are less wellunderstood.

Many methods may be used to introduce the live attenuated virusformulations to a human or animal subject to induce an immune response;these include, but are not limited to, oral, intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous and intranasal routes. It ispreferable to introduce the chimeric virus vaccine via its natural routeof infection.

Any virus may be engineered in accordance with the invention to producean attenuated strain suitable for use as a safe live-virus vaccine,including but not limited to viruses belonging to the families set forthin Table I below.

TABLE I FAMILIES OF HUMAN AND ANIMAL VIRUSES VIRUS CHARACTERISTICS VIRUSFAMILY dsDNA Enveloped Poxviridae Irididoviridae HerpesviridaeNonenveloped Adenoviridae Papovaviridae Hepadnaviridae ssDNANonenveloped Parvoviridae dsRNA Nonenveloped Reoviridae BirnaviridaesRNA Enveloped Positive-Sense Genome No DNA Step in ReplicationTogaviridae Flaviviridae Coronaviridae Hepatitis C Virus DNA Step inReplication Retroviridae Negative-Sense Genome Non-Segmented GenomeParamyxoviridae Rhabdoviridae Filoviridae Segmented GenomeOrthoinyxoviridae Bunyaviridae Arenaviridae Nonenveloped PicornaviridaeCalciviridae Abbreviations used: ds = double stranded; ss = singlestranded; enveloped = possessing an outer lipid bilayer derived from thehost cell membrane; positive-sense genome = for RNA viruses, genomesthat are composed of nucleotide sequences that are directly translatedon ribosomes, = for DNA viruses, genomes that are composed of nucleotidesequences that are the same as the mRNA; negative-sense genome = genomesthat are composed of nucleotide sequences complementary # to thepositive-sense strand.

DNA viruses (e.g., vaccinia, adenoviruses, baculovirus) and positivestrand RNA viruses (e.g., poliovirus) may be readily engineered usingrecombinant DNA techniques which are well known in the art (e.g., seeU.S. Pat. No. 4,769,330 to Paoletti; U.S. Pat. No. 4,215,051 to Smith;Racaniello et al., 1981, Science 214: 916-919). Until recently, however,negative strand RNA viruses (e.g., influenza) were not amenable to sitespecific genetic manipulation because the viral RNAs are not infectious.However, a recently developed technique, called “reverse genetics,”allows the engineering and production of recombinant negative strand RNAviruses.

The reverse genetics technique involves the preparation of syntheticrecombinant viral RNAs that contain the non-coding regions of thenegative strand virus which are essential for the recognition of viralRNA by viral polymerases and for packaging signals necessary to generatea mature virion. The recombinant RNAs are synthesized from a recombinantDNA template and reconstituted in vitro with purified viral polymerasecomplex to form recombinant ribonucleoproteins (RNPs) which can be usedto transfect cells. A more efficient transfection is achieved if theviral polymerase proteins are present during in vitro transcription ofthe synthetic RNAS. The synthetic recombinant RNPs can be rescued intoinfectious virus particles. The foregoing techniques are described inU.S. Pat. No. 5,166,057 and in Enami & Palese, 1991, J. Virol. 65:2711-2713, each of which is incorporated by reference herein in itsentirety), and influenza A viruses containing insertions, deletions andmutations with the stalk portion of the NA gene, one of which changesacts as a host range mutant. Using the reverse genetics technique, thefollowing recombinant negative-strand viruses were engineered: aninfluenza virus containing nine different RNA segments; a chimericinfluenza A virus (NA/B-NS) in which the noncoding region of the NA genewas replaced by that belonging to an influenza B virus NS gene; aninfluenza A virus with chimeric hemagglutinins containing epitopes fromdifferent influenza subtypes (Enami et al., 1991, Virology 185: 291-298;Muster et al., 1991, Proc. Natl. Acad. Sci. USA 88: 5711-5781; copendingapplication Ser. No. 07/841,310 filed Feb. 3, 1992; and Li et al., 1992,J. Virol. 66: 399-404; each of which is incorporated by reference hereinin its entirety); and influenza A viruses containing insertions,deletions, and mutations within the stalk portion of the NA gene, one ofwhich acts as a host range mutant. The invention is discussed in moredetail in the subsections below and the examples infra. For clarity, thedetails of the invention are described using influenza. However, theprinciples may be analogously applied to construct other attenuatedviruses.

5.1. DOWN-REGULATION OF VIRAL GENES

In accordance with the invention, a non-coding regulatory region of avirus can be altered to down-regulate any viral gene, e.g. reducetranscription of its mRNA and/or reduce replication of vRNA (viral RNA),so that an attenuated virus is produced.

This approach, while applicable to any virus, is particularly attractivefor engineering viruses with segmented genomes; i.e., viruses in whichthe genome is divided into segments that are packaged into virions. Forexample, the segmented genome of influenza A virus (an orthomyxovirus)consists of eight molecules of linear negative-sense ssRNAs which encodeten polypeptides, including: the RNA-directed RNA polymerase proteins(PB2, PB1 and PA) and nucleoprotein (NP) which form the nucleocapsid;two surface glycoproteins which project from the envelope: hemagglutinin(HA) and neuraminidase (NA); and nonstructural proteins (NS1 and NS2)whose function is unknown. The termini of each segment contain thenon-coding regions essential for recognition by viral polymerase and forpackaging signals necessary to generate a mature virion. The sequence ofthe termini is highly conserved among all eight segments. As anotherexample, the segmented genome of reoviruses consists of 10 to 12segments of linear dsRNA which encode 6 to 10 major structuralpolypeptides, a transcriptase and other enzymes.

Alterations of non-coding regulatory regions of segmented viruses whichresult in down-regulation of replication of a viral gene segment, and/ordown-regulation of transcription of a viral gene will result in theproduction of defective particles in each round of replication; i.e.particles which package less than the full complement of viral segmentsrequired for a fully infectious, pathogenic virus. Therefore, thealtered virus will demonstrate attenuated characteristics in that thevirus will shed more defective particles than wild type particles ineach round of replication. However, since the amount of proteinsynthesized in each round is similar for both wild type virus and thedefective particles, such attenuated viruses are capable of inducing agood immune response.

The foregoing approach is equally applicable to non-segmented viruses,where the down regulation of transcription of a viral gene will reducethe production of its mRNA and the encoded gene product. Where the viralgene encodes a structural protein, e.g., a capsid, matrix, surface orenvelope protein, the number of particles produced during replicationwill be reduced so that the altered virus demonstrates attenuatedcharacteristics; e.g., a titer which results in subclinical levels ofinfection. For example, a decrease in viral capsid expression willreduce the number of nucleocapsids packaged during replication, whereasa decrease in expression of the envelope protein may reduce the numberand/or infectivity of progeny virions. Alternatively, a decrease inexpression of the viral enzymes required for replication, e.g., thepolymerase, replicase, helicase, and the like, should decrease thenumber of progeny genomes generated during replication. Since the numberof infectious particles produced during replication are reduced, thealtered viruses demonstrate attenuated characteristics. However, thenumber of antigenic virus particles produced will be sufficient toinduce a vigorous immune response.

Any alteration of the regulatory non-coding regions which decrease theirefficiency or strength may be engineered in accordance in the invention.For example, the strength of viral promoters can be reduced byalterations in the stem structure. In the experiments detailed herein,specific nucleotide changes in the second stem structure of the promoter(B/E in FIG. 1) at the termini of the vRNAs which make up the panhandlestructure of segmented negative-strand strand RNA viruses, such asinfluenza, were found to be responsible for the down-regulation of thesynthesis of one vRNA segment. In particular, the UCCU/AGGA nucleotidesof the chimeric influenza mutant NA/DeF/AbC (Section 7.2.1, Table III)are the critical base pairs involved. (The comparable base pairs in thewild type are CUC/GAG).

This base pair combination was introduced into the non-coding regulatoryregions for other viral genes. Results indicate that the chimericviruses so produced are also attenuated. Apparently, changes in thissecond stem structure lead to a reduction in vRNA synthesis of the viralsegment, which is accompanied by a reduction in the number of infectiousparticles containing a full complement of all eight RNA segments. Itshould also be noted that the reversion rate of the changed stemstructure is extremely low, since reversion would require thesimultaneous change of two nucleotides.

While the engineered reduction in vRNA or mRNA produces attenuation,this is not accompanied by a significant reduction in the viral proteinspecified by the gene. In fact, it may be desirable to engineer strongtranslation signals into the viral gene so that the gene transcriptswhich are present at low concentrations are efficiently translated intoviral proteins required to provide some degree of replication.

The experiments described in the examples detailed infra were designedto understand the molecular mechanism underlying the changed growthcharacteristics of the chimeric NA/B-NS virus, as well as otherengineered viruses described herein (e.g., RAM 3, HA/malaria ME 1, andHA/poliovirus 1) with the aim of developing a prototype for live virusvaccines. The NA/B-NS is a transfectant influenza A virus containing achimeric NA gene in which the noncoding sequences are identical to thosein the NS gene of influenza B/Lee virus described in copendingapplication Ser. No. 07/841,310, filed Feb. 3, 1992 now abandoned, byPalese et al.; and in Muster et al., 1991, Proc. Natl. Acad. Sci. USA88: 5177-5181 each of which is incorporated by reference herein in itsentirety). This virus has many unique growth characteristics in tissueculture and it is highly attenuated in mice.

Several lines of evidence obtained from the experiments described hereinindicate that the cis elements derived from the influenza B/Lee virusgene are responsible for the dramatic effects on transcription andreplication of the chimeric NA gene of the NA/B-NS transfectant virus.It was found that the NA gene had a six-fold lower representation in thepurified viral preparation than did the remaining seven RNAs (seeSections 6.2.2 and 6.2.4, infra). This strikingly lower representationof one RNA is compatible with the finding that the NA/B-NS transfectantvirus has an approximately 5- to 10-fold lower infectious particle tophysical particle ratio than wild type virus (see Section 6.2.1, infra).It is assumed that an infectious virus would require the presence of afull complement of all eight influenza virus RNA segments. Many of theNA/B-NS progeny virus, however, lack an NA gene, so that more defectiveparticles are formed than is the case in a wild type virus infection. Itis not clear whether this 5- to 10-fold reduction in titer is only thereflection of the lower representation of the NA gene or whether otherfactors also play a role. For example, some viruses may containdefective interfering RNAs which would lower the infectivity titer ofthe preparation.

The mRNA synthesis of the NA is also considerably reduced intransfectant virus-infected cells (see Section 6.2.4, infra).Surprisingly, this does not lead to a commensurate reduction in proteinsynthesis. Both virus-infected cells and purified virus show only atwo-fold lower level of NA protein relative to that of wildtype-infected cells or of purified wild type virus itself (see Section6.2.3, infra). This higher than expected level of NA protein in thetransfectant virus may be the result of a good Kozak sequence present inthe chimeric NA gene. The chimeric NA gene has an A in the −3 positioninstead of the U found in the wild type NA RNA (see open triangle inFIG. 1). The data also indicate that the two-fold reduction in NAactivity does not significantly influence the pathogenicity of thevirus, since other transfectant viruses were constructed (e.g., NAM 3shown below) in which the expression of the NA gene is down regulated bya factor of 10 without affecting virus growth in tissue culture (seeSection 7.2.2 and Table IV, infra):

   AG.A     A         UUUGAACAAACAUU AGU    AAC.. AGGAGUUUUCG    UUG   UCCUCAAA    ...U    CG         CUUAC

It should be noted that in the transfectant virus, expression of theother seven genes is not altered. Specifically, the NA/B-NS virusproduces the same level of HA protein as found in wild type virus andcomparable amounts of HA are packaged into the envelope of the virus. Itappears that the attenuation characteristic is best explained by thelower synthesis of NA-specific vRNA and by the resulting lowerrepresentation of particles containing a full complement of the eightRNA segments.

How influenza A virus packages its eight RNA genome segments remains aninteresting question. In the past, two different mechanisms wereproposed for the packaging of influenza virus RNAs: one suggests thatthe eight RNAs are selectively packaged and the other that viral RNAsare packaged randomly (Compans et al., 1970, In The Biology Of Large RNAViruses, Barry & Mahy, Eds., pp. 87-108, Academic Press, New York; Lamb& Choppin, 1983, Ann. Rev. Biochem. 467-506; Smith & Hay, 1982, Virology118: 96-108). Evidence is now accumulating to support the randompackaging mechanism. The random packaging theory originated from thefact that influenza viruses have a low ratio of infectious particles tophysical particles. If one assumes that an average of 11 RNAs arepackaged per virion, the expected ratio is compatible with that found invivo (Enami et al., 1991, Virology 185: 291-298). This model was alsosupported by the finding of a reassortant virus which contained twocopies of the same segment derived from two different viruses(Scholtissek, 1978, Virology 89: 506-516), and further support for thistheory came from a more recent report which described an influenza Avirus which required nine RNAs in order to be infectious (Enami et al.,1991, Virology 185: 291-298). The data described in the examples, infra,concerning the characterization of the NA/B-NS transfectant virus alsoseem to favor the random packaging mechanism rather than the selectiveone. The lower level of chimeric NA RNA found in virions is consistentwith the reduction of its synthesis in infected cells. In a selectivepackaging model, it would be expected that approximately equimolaramounts of chimeric NA RNA would be packaged into virus particles.

In summary, the experiments described infra, indicate that influenzaviruses for which the synthesis of a vRNA segment is down-regulatedproduce defective particles during replication. Since the proteins ofthis virus are unaltered as compared to wild type virus, attenuationmust be the result of inefficient cis-acting signals. This principal ofattenuation may be applied analogously to other viruses with segmentedgenomes. For example, the introduction of modifications into thenoncoding sequences of rotavirus genes or of genes of other segmenteddsRNA viruses (Roner et al., 1990, Virology 179: 845-852) should alsoallow the pathogenicity of these viruses to be altered.

5.2. ALTERATION OF VIRAL PROTEINS

An alternative way to engineer attenuated viruses involves theintroduction of an alteration, including but not limited to aninsertion, deletion or substitution of one or more amino acid residuesand/or epitopes into one or more of the viral proteins. This may bereadily accomplished by engineering the appropriate alteration into thecorresponding viral gene sequence. Any change that alters the activityof the viral protein so that viral replication is modified or reducedmay be accomplished in accordance with the invention.

For example, alterations that interfere with but do not completelyabolish viral attachment to host cell receptors and ensuing infectioncan be engineered into viral surface antigens or viral proteasesinvolved in processing to produce an attenuated strain. According tothis embodiment, viral surface antigens can be modified to containinsertions, substitution or deletions of one or more amino acids orepitopes that interfere with or reduce the binding affinity of the viralantigen for the host cell receptors. This approach offers an addedadvantage in that a chimeric virus which expresses a foreign epitope maybe produced which also demonstrates attenuated characteristics. Suchviruses are ideal candidates for use as live recombinant vaccines. Forexample, heterologous gene sequences that can be engineered into thechimeric viruses of the invention include, but are not limited to,epitopes of human immunodeficiency virus (HIV) such as gp120; hepatitisB virus surface antigen (HBsAg); the glycoproteins of herpes virus(e.g., gD, gE); VP1 of poliovirus; and antigenic determinants ofnonviral pathogens such as bacteria and parasites to name but a few.

In this regard, influenza is an ideal system in which to engineerforeign epitopes, because the ability to select from thousands ofinfluenza virus variants for constructing chimeric viruses obviates theproblem of host resistance or immune tolerance encountered when usingother virus vectors such as vaccinia. In addition, since influenzastimulates a vigorous secretory and cytotoxic T cell response, thepresentation of foreign epitopes in the influenza background may alsoprovide for the secretory immunity and cell-mediated immunity. By way ofexample, the insertion, deletion or substitution of amino acid residuesin the HA protein of influenza can be engineered to produce anattenuated strain. In this regard, alterations to the B region or Eregion of HA may be utilized. In accordance with this approach, themalarial epitope (ME 1) of Plasmodium yoelii (NEDSYVPSAEQI) (SEQ IDNO: 1) was introduced into the antigenic site E of the hemagglutinin ofinfluenza. The resulting chimeric virus has a 500- to 1,000-fold lowerLD₅₀ (lethal dose 50) than that of wild type virus when assayed in mice.In another embodiment, the major antigenic determinant of poliovirustype 1, i.e., the BC loop of the VP1 of poliovirus type 1 (PASTTNKDKL)(SEQ ID NO: 4) was engineered into the B region of the influenza HAprotein. This chimeric virus is also attenuated (e.g., see Section 8,infra).

In another embodiment, alterations of viral proteases required forprocessing viral proteins can be engineered to produce attenuation.Alterations which affect enzyme activity and render the enzyme lessefficient in processing, should affect viral infectivity, packaging,and/or release to produce an attenuated virus. For example, alterationsto the NA protein of influenza can be engineered to reduce NA enzymeactivity and decrease the number and/or infectivity of progeny virusreleased during replication. The example presented in Section 9describes the production of an influenza A recombinant virus containinga deletion in the stalk region of the NA gene. This mutant acts as ahost range mutant, i.e., a virus that has lost the capability to inflectcertain cell types. In another example, the protease of togaviruses,flaviviruses or hepatitis C virus (HCV) could be altered so thatappropriate: cleavage of the viral polyprotein is reduced, resulting ina decrease in the number of progeny virions produced during replication.

In another embodiment, viral enzymes involved in viral replication andtranscription of viral genes, e.g., viral polymerases, replicases,helicases, etc. may be altered so that the enzyme is less efficient oractive. Reduction in such enzyme activity may result in the productionof fewer progeny genomes and/or viral transcripts so that fewerinfectious particles are produced during replication.

The alterations engineered into any of the viral enzymes include but arenot limited to insertions, deletions and substitutions in the amino acidsequence of the active site of the molecule. For example, the bindingsite of the enzyme could be altered so that its binding affinity forsubstrate is reduced, and as a result, the enzyme is less specificand/or efficient. For example, a target of choice is the viralpolymerase complex since temperature sensitive mutations exist in allpolymerase proteins. Thus, changes introduced into the amino acidpositions associated with such temperature sensitivity can be engineeredinto the viral polymerase gene so that an attenuated strain is produced.

6. EXAMPLE: REDUCED TRANSCRIPTION AND REPLICATION OF NA GENE OFINFLUENZA NA/B-NS IS RESPONSIBLE FOR ATTENUATION

In order to understand the molecular characteristics responsible for theattenuation of the transfectant virus, NA/B-NS, the following series ofexperiments were carried out to analyze the virus at the molecularlevel. The data presented in the subsections below indicate thatattenuation results from the effect of the altered cis elements on thereplication of the chimeric NA gene. A low level of the NA gene in thevirus preparation results in a higher proportion of defective particlesthan is found in wild type virus preparation. In addition, the datasupport a random mechanism for the packaging of vRNAs into influenzavirus particles.

6.1. MATERIALS AND METHODS 6.1.1. VIRUSES AND CELLS

The stocks of influenza A/WSN/33 virus and NA/B-NS transfectant virus(Muster et al., 1991, Proc. Natl. Acad. Sci. USA 88: 5177-5181) wereprepared from purified plaques by growing them in Madin-Darby bovinekidney (MDBK) cells in reinforced MEM medium (REM) containing 2 μg/mltrypsin. MDBK cells were used for the plaguing of viruses and the studyof virus specific RNA synthesis.

6.1.2. PLASMIDS

In order to generate riboprobes, several plasmids were constructed.pSP64-NS DNA contains the entire NS segment of A/WSN/33 virus insertedinto the Sal I site of the pSP64 vector in the orientation by which mRNAsense NS RNA can be made using SP6 RNA polymerase. The fragment derivedfrom pSP64-NS by digestion with Hind III and Eco RI was inserted betweenthe Hind III and the Eco RI site of the IBI30 vector. The resultingplasmid IBI30-NS can produce minus sense NS RNA using T7 RNA polymerase.In order to obtain plasmid ΔT3NAv which produces minus sense NA specificRNA probe, the DNA in the pT3NAv plasmid (Enami et al., 1990, Proc.Natl. Acad. Sci. USA 87: 3802-3805) was shortened by deleting thefragment between the Bam HI and the Eco RI sites. In addition, wecreated a plasmid designated IBI30-NA by inserting the fragment betweenthe Xba I and Eco RI sites of pT3NAv into the IBI30 vector. This plasmidcan generate positive sense NA specific RNA by T7 polymerasetranscription.

6.1.3. VIRUS PURIFICATION AND RNA EXTRACTION

Influenza A/WSN/33 virus and NA/B-NS transfectant virus were grown inMDBK cells, and then purified through 30-60% sucrose gradientcentrifugation as described previously (Enami et al., 1990, Proc. Natl.Acad. Sci. USA 87: 3802-3805). Virus purified from four 175 cm² flasksof MDBK cells was resuspended in 0.3 ml TMK buffer (10 Mm Tris, pH 7.5,1.5 mM MgCl₂, 10 mM KCl) and disrupted by incubation with 9 μl 10% SDSand 7.5 μl proteinase K (10 mg/ml) at 56° C. for 10 minutes, followed byaddition of 35 μl SLN buffer (5% SDS, 1.4 M LiCl, 100 mM NaOAC, pH 7.0).Virion RNAs were extracted with phenol-chloroform and collected byethanol precipitation. For isolating viral RNAs from infected cells,MDBK cells were infected with either influenza A/WSN/33 virus or NA/B-NStransfectant virus at an m.o.i.=1, and harvested at the indicated timepoints. Cells were washed twice with ice-cold PBS and lysed with 4 Mguanidinium isothiocyanate (Sigma). The total RNA was then purified byequilibrium centrifugation in 5.7 M cesium chloride (Fisher) (Luo etal., 1991, J. Virol. 65: 2861-2867).

6.1.4. DETERMINATION OF THE RATIO OF INFECTIOUS AND PHYSICAL PARTICLES

In order to determine the total number of physical particles ofinfluenza A/WSN/33 virus and NA/B-NS transfectant virus, the viruspreparations were mixed with an equal volume of a suspension ofcarboxylate polystyrene beads (0.1μin diameter) at a concentration of4.5×10⁹ particles per milliliter (Polyscience, Inc., Warrington, Pa.),and then stained with phosphotungstic acid. The ratio of virus andpolystyrene beads was determined by counting the two different particlesunder the electron microscope. For measuring the number of infectiousparticles in the preparations, the virus stocks were serially dilutedand plaqued in MDBK cells.

6.1.5. RNA ELECTROPHORESIS

RNAs extracted from influenza A/WSN/33 virus and NA/B-NS transfectantvirus were electrophoresed on a 3% polyacrylamide gel containing 7.7 Murea at 150 volts for two hours. The RNA segments were visualized bysilver staining as described previously (Enami et al., 1990, Proc. Natl.Acad. Sci. USA 87: 3802-3805).

6.1.6. RIBONUCLEASE PROTECTION ASSAY

Ribonuclease protection assay (RPA) was used for quantitation of virionRNA in viral particles as well as for the measurement of viral mRNAs andcRNAs in infected MDBK cells. The NS segment was chosen as an internalcontrol. For measuring virion RNAs, positive sense NS and NA specificRNA probes were generated by run-off transcription using phase SP6 or T7RNA polymerase and plasmid PSP64-NS DNA linearized by Nco I and plasmidIBI30-NA digested with Fok I, respectively. To determine the amount ofmRNAs and cRNAs, minus sense NS and NA specific RNA probes weretranscribed from Dde I digested IBI30-NS DNA and Pvu II cut ΔT3NA DNA,respectively, using phage T7 or T3 RNA polymerase respectively. The RNAprobes were labelled with α³²P[UTP] (800 Ci/mM, Du Pont, NEN, Boston,Mass.). In general, 50 ng of virion RNA extracted from purified viruswas hybridized to 5×10⁴ cpm each of positive sense NS and NA specificprobes, or 5 μg of total RNA isolated from virus-infected cells washybridized to 5×10⁴ cpm each of minus sense NS and NA specific probes.After 12 hours incubation at 45° C., the hybridization mixture wasdigested by RNase A/T₁, following the manufacturer's instruction (AmbionInc., Austin, Tex.), and the resulting products were analyzed on a 6%acrylamide denaturing gel (Luo et al., 1990, J. Virol. 64: 4321-4328).

6.1.7. PRIMER EXTENSION

The genomic RNAs (vRNAs) of NA and NS segments of influenza A/WSN/33virus and of NA/B-NS transfectant virus were quantitated by primerextension (Luo & Taylor, 1990, J. Virol. 64: 4321-4328). The primers fordetecting NS and NA specific vRNA are 21 nt long and are complementaryto minus sense vRNA. The NS primer, 5′-GGGAACAATTAGGTCAGAAGT-3′(SEQ IDNO: 2) spans the region between nucleotides 695 to 714 of the NS cRNA.The NA primer, 5′-GTGGCAATAACTAATCGGTCA-3′(SEQ ID NO: 3) coves thenucleotides 1151 to 1171 of the NA cRNA. Both NS and NA primers were5′-end labelled by incubation with ^(γ32)P-ATP (3000 Ci/mM, Du Pont,NEN, Boston, Mass.) and T4 DNA kinase (Biolabs, Beverly, Mass.). 100 ngof RNA extracted from virus or 5 μg of total RNA isolated from infectedMDBK cells was reverse transcribed by RNase H minus MLV reversetranscriptase (BRL, Gaithersburg, Md.) in the presence of 3×10⁵ cpm ofeach of 5′-end labelled NS and NA primers. After incubation at 37° C.for 2 hours, the reaction was stopped by addition of EDTA to 10 mM, andfollowed by phenol-chloroform extraction and alkali treatment (Luo &Taylor, 1990, J. Virol. 64: 4321-4328). The products were analyzed on a6% polyacrylamide gel containing 7 M urea. The amount of product wasmeasured by directly counting the radioactivity of the gel piececorresponding to each band on the film.

6.1.8. NEURAMINIDASE ASSAY AND WESTERN ANALYSIS

For the assay of neuraminidase activity of influenza A/WSN/33 andNA/B-NS transfectant viruses,2′-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (Sigma) was usedas substrate. The reaction mixture consisted of 25 μl 2 mM substrate, 25μl virus and 50 μl 0.2 M phosphate buffer (pH 6.0) containing 2 mMCaCl₂. After incubation at 37° C. for 10 minutes, the reaction wasstopped by addition of 2 ml 0.5 M glycine-NaOH buffer (pH 10.6), andthen the neuraminidase activity was determined by measuring thefluorescence with excitation at 365 nm and emission at 450 nm, usingmethylumbeliferone as a standard. The protein concentration of theviruses was measured using the Bio-Rad protein assay kit. For theWestern analysis of NA and HA proteins of WSN/33 and NA/B-NS viruses,viral proteins were electrophoresed on a 10% Laemmli gel (Laemmli, 1970,Nature 227: 680-685), and subsequently transferred to a nitrocellulosemembrane (Schleicher & Schuell, Keene, N.H.). A monoclonal antibodydirected against carbohydrates was used to detect the NA and HAglycoproteins of the viruses. The Western blot was developed with a ratantibody against mouse kappa chains which was labelled with ¹²⁵Iodine.

6.1.9. ANALYSIS OF PROTEIN SYNTHESIS IN INFECTED CELLS

MDBK cells (35 mm dish) were infected with either WSN/33 virus orNA/B-NS transfectant virus at an moi of approximately 3. Thismultiplicity was used because the NA/B-NS transfectant virus did notgrow to higher titer. At indicated times, the proteins were labelled incysteine free medium with [³⁵S] cysteine (1027 Ci/mmol, Du Pont, NENResearch Products) at 100 μci/ml medium for 30 minutes. The cells werethen washed twice with ice-cold PBS buffer and lysed in 150 μl lysisbuffer containing 1% NP-40, 150 Mm NaCl, 50 mM Tris-HCl pH 8.0, and 1 mMPMSF. About {fraction (1/20)}th of the sample was loaded onto a 10%Laemmli gel (Laemmli, 1970, Nature 227: 680-685).

6.2. RESULTS

The results described below indicate that influenza viruses, for whichthe synthesis of a vRNA segment is down-regulated, produce defectiveparticles during replication. Since the proteins of the NA/B-NS virusare unaltered as compared to wild type virus, attenuation must be theresult of inefficient cis-acting signals.

6.2.1. RATIO OF INFECTIOUS TO PHYSICAL PARTICLES

The genome of the transfectant NA/B-NS virus differs from wild typeinfluenza A/WSN/33 virus only in the noncoding region of the NA gene. Itis thus likely that the altered biological properties of thetransfectant virus are the result of altered cis signals located in thenoncoding region of the chimeric NA gene. Specifically, it was notedearlier that the transfectant virus grew to lower titers than wild typevirus in MDBK cells, MDCK cells and in mice. In addition, the lowmultiplicity growth curves in tissue culture were significantly delayedrelative to those of wild type virus (Muster et al., 1991, Proc. Natl.Acad. Sci. USA 88: 5177-5181). The transfectant virus was examined for atemperature sensitive phenotype, which could explain the altered growthcharacteristics. However, the pattern of the growth curve at 30° C., 33°C., 37° C. and 38° C. for NA/B-NS virus was not different from that ofwild type virus at the corresponding temperatures. To answer thequestion of whether defective particles were present in the NA/B-NSvirus preparation, the virus was characterized by counting the physicalparticles under the electron microscope and comparing this number withthe plaque-forming units of the preparation. Interestingly, the NA/NS-Btransfectant virus showed a similar number of physical particles as wildtype virus, but consistently lower PFU titers (Muster et al., 1991,Proc. Natl. Acad. Sci. USA 88: 5177-5181) (Table II).

TABLE II The ratio of infectious particles to physical particles ofinfluenza A/WSN/33 virus and NA/B-NS transfectant virus. No. of physicalNo. of infectious Ratio Viruses particles (pp) particles (ip) pp/ipWSN/33 1.8 × 10⁹/ml 1.2 × 10⁸/ml 15 NA/B-NS 1.2 × 10⁹/ml 1.0 × 10⁷/ml120

Thus, the NA/B-NS virus grown in MDBK cells shows at least a 5 to10-fold lower infectious particle to physical particle ratio than isseen with the WSN/33 wild type virus.

6.2.2. CHARACTERIZATION OF THE RNA OF THE ATTENUATED VIRUS

Since the NA/B-NS virus contains many defective particles, the viral RNAwas examined for the presence of defective RNAs. Following extractionfrom purified virus, the genomic RNA was separated on a 3%polyacrylamide gel containing 7.7m urea. As shown in FIG. 2A, thechimeric NA RNA of the NA/B-NS transfectant virus is almost invisible onthe gel, whereas the other seven segments are present in approximatelyequimolar concentrations. When the amount of RNA was increased on thegel, the chimeric NA RNA can be shown to migrate at the same position asthe control RNA in FIG. 2A, lane 3. However, electrophoresis and silverstaining did not permit the quantitation of the chimeric NA RNA packagedin virions. For this purpose, the ribonuclease protection assay (RPA)and primer extension experiments were performed. Positive senseNS-specific and NA-specific probes were hybridized in the same reactionto purified virion RNA from either WSN/33 virus (FIG. 2A, lane 2) orNA/B-NS transfectant virus (FIG. 2A, lane 3) and then digested by RNaseA/T₁. The resulting products were analyzed on a 6% polyacrylamide gelcontaining 7M urea (Luo et al., 1991, J. Virol. 65: 2861-2867), and theamounts of vRNA were calculated by counting the radioactivity of the gelslices corresponding to the bands on the film. As shown in FIG. 2B, theprobe protected by the chimeric NA RNA migrates faster than that of thewild type gene because the 5′-noncoding sequences are different in thetwo NA genes. When compared to the NA segment of WSN/33 virus, theamount of chimeric NA RNA in the transfectant virus is about 6 timeslower using the NS gene as an internal control. The primer extensionexperiment, as shown in FIG. 2C, shows similar reduction of the chimericNA RNA packaged in virions relative to the NS gene, suggesting aspecific lower representation of the chimeric NA gene in thetransfectant virus preparation.

6.2.3. CHARACTERIZATION OF THE PROTEIN OF THE ATTENUATED VIRUS

To determine whether or not the NA/B-NS transfectant virus alsocontained less neuraminidase protein, neuraminidase assays and a Westernanalysis of the NA protein in virus particles were conducted. For theenzymatic assay of the neuraminidase, the total concentration of viralproteins was first determined by protein assay, and then the same amountof purified virus was used. The NA/B-NS transfectant virus showed only atwo-fold lower NA activity than the WSN/33 wild type virus. A similarfinding was obtained by Western analysis, which showed 1.9 fold less NAprotein, but the same amount of HA protein in virions of the NA/B-NStransfectant virus as compared to those of WSN/33 virus (FIG. 5A).

6.2.4. VIRUS-SPECIFIC RNA SYNTHESIS IN INFECTED CELLS

Based on the vRNA analysis of the NA/B-NS transfectant virus, there islittle doubt that less chimeric NA RNA is contained in the viralparticles. The lower level of the chimeric NA RNA in the virions couldbe caused either by a change in the packaging signal leading to lessefficient packaging of the NA RNA or by a change in the synthesis ofgenomic NA RNA which could be the result of inefficient recognition ofthe influenza B virus specific promoter by the influenza A viralpolymerase. To pinpoint the exact mechanism, the synthesis of thechimeric NA gene in infected MDBK cells was examined. Considering thatvRNA is mainly synthesized in the late phase of infection (Shapiro etal., 1987, J. Virol. 61: 764-773), RNA was extracted from cells at 7,8½, and 10 hours post-infection. Five μg of total RNA extracted fromvirus-infected cells was used for the primer extension analysis. Thedata in FIG. 4 show that vRNA synthesis of the chimeric NA gene wasremarkably decreased relative to that of control virus. When compared tothe synthesis of the NA RNA of WSN/33 virus, the chimeric NA gene isreduced by a factor of 9, 8, and 8 at 7, 8½, and 10 hours p.i.,respectively. However, no reduction of vRNA synthesis was observed withrespect to the NS segment of the NA/B-NS transfectant virus. This resultindicates that the reduction of the chimeric NA RNA in transfectantvirus is the result of its lower synthesis in cells rather than due to adefect in the packaging of the chimeric NA RNA.

In order to determine the level of mRNA synthesis of the chimeric NAgene, MDBK cells were infected with either WSN/33 virus or NA/B-NStransfected virus, and total RNA was then isolated from virus-infectedcells at different times post-infection. Subsequently, the level ofvirus specific mRNAs was quantitated by RPA. Again the NS segment wasused as control (see FIGS. 3A-3C). From the time course, it is apparentthat the level of chimeric NA mRNA is markedly reduced relative to thatof the wild type virus and that it only increases slightly with time. At5 hours p.i., the level of chimeric NA mRNA was five-fold less than thatof WSN/33 virus (FIG. 3C), whereas the NS-specific mRNA synthesis of theNA/B-NS transfectant virus was similar to that of WSN/33 virus at theindicated times (FIG. 3B). It should be noted that the NS-specific mRNAsynthesis appears earlier and is more efficient than NA-specific mRNAsynthesis in both WSN/33 and NA/B-NS transfectant virus infected cells(probes of similar activity were used in the experiment). This findingis in good agreement with previous reports (Enami et al., 1985, Virology142: 68-77), and suggests that the mRNA synthesis of different influenzavirus RNA segments is differentially regulated. The conditions of theassay did not permit quantitation of the level of cRNA of the chimericNA gene because the cRNA synthesis was found to be only 3-5% of that ofmRNA synthesis.

6.2.5. REDUCTION OF NA PROTEIN SYNTHESIS IN VIVO

The NA protein was found to be reduced by a factor of two in virions ofthe NA/B-NS transfectant virus, whereas the synthesis of chimeric NAmRNA was reduced by much more. The question then arose whether the NAprotein encoded by the chimeric NA mRNA inside cells parallels theamount of its mRNA, or whether there is a selective incorporation of theneuraminidase into the viral envelope. In order to answer this question,the synthesis of NA protein in infected cells was measured. Viralproteins were labelled with ³⁵S[cysteine] at 1, 3, 5, and 7 hourspost-infection. The cell lysates were then analyzed on a 10% Laemmli gel(Laemmli, 1970, Nature 227: 680-685) and the proteins were quantitatedby an AMBIS radioanalytic imaging system (Luo & Taylor, 1990, J. Virol.64: 4321-4328). As shown in FIG. 5B, synthesis of the NA protein ofNA/B-NS transfectant virus was two times lower than that of wild typevirus at both 5 and 7 hrs p.i. From this experiment, it can be concludedthat the assembly of the NA protein into virions parallels its synthesisin infected cells.

7. EXAMPLE: PANHANDLE BASE PAIRS INVOLVED IN ATTENUATION OF INFLUENZAVIRUS

The experiments described below were designed to identify the nucleotidesequences responsible for attenuation and the effects of gene expressionon viral replication.

7.1. MATERIALS AND METHODS

The reverse genetics technique was used to engineer mutations into thepanhandle structure of the NA segment of influenza. Viruses and cellswere prepared, purified and analyzed for attenuation as described inSection 6.1 and its subsections, supra.

7.2. RESULTS 7.2.1. ATTENUATION SEQUENCE IN THE SECOND STEM STRUCTURE OFTHE VIRAL PROMOTER

A series of chimeric mutants constructed and analyzed for attenuationare shown in Table III below. The results indicate that UCCU/AGGA of theNA/DeF/AbC influenza mutant are the critical base pairs involved inattenuation.

7.2.2. REDUCTION IN PROTEIN AND ENZYME ACTIVITY

An additional attenuated mutant, designated NAM-3 has the followingstructure:

   AG.A     A         UUUGAACAAACAUU AGU    AAC.. AGGAGUUUUCG    UUG   UCCUCAAA    ...U    CG         CUUAC

NAM 3, like NA/B-NS, demonstrates attenuation in that defectiveparticles are produced during each round of replication. However, NAM 3also demonstrated a ten-fold decrease in neuraminidase expression andenzyme activity (Table IV). This decrease in expression of the enzymedid not affect viral replication.

TABLE IV NA EXPRESSION AND ACTIVITY IN INFLUENZA VIRUS MUTANT EnzymeActivity² Strain Protein¹ 10 min 20 min 40 min WSN-wt 1.00 3.6 5.48 5.17NAM 3 0.14 0.19 0.26 0.67 ²Relative intensities of NA bands displayed onSDS-PAGE of infected cells. See Section 6.1.9 for Methods. ²Virus waspurified over sucrose containing Ca⁺⁺ as described in Section 6.1.3.Enzyme activity was assayed described in Section 6.1.8, and is expressedas percent substrate conversion by 1 ng virus/minute.

8. EXAMPLE: EXPRESSION OF FOREIGN EPITOPES IN THE ANTIGENIC SITES B OR EOF HA OF INFLUENZA RESULTS IN ATTENUATION

The experiments described below indicate that alterations to the B or Eregion of HA may be engineered to construct attenuated chimericinfluenza viruses.

8.1. MATERIALS AND METHODS

The reverse genetics techniques described in Section 6.1.1. supra, wereutilized to (a) introduce the malarial epitope (ME 1) of Plasmodiumyoelii, (NEDSYVPSAEQI) into antigenic site E of the HA of influenza (seeFIG. 6A); or (b) to introduce the major antigenic determinant ofpoliovirus type 1, i.e. the BC loop of the VP1 of poliovirus type 1(PASTTNKDKL), into antigenic site B of HA of influenza (see FIG. 6B).The LD₅₀ of chimeric and wild type influenza was determined in mice bythe Karber method (see Muster et al., 1991, Proc. Natl. Acad. Sci. USA88: 5177-5181).

8.2. RESULTS

The chimeric influenza/malaria virus had a 500- to 1000-fold lower LD₅₀than that of wild type virus when assayed in mice. Likewise the chimericinfluenza/polio virus demonstrated a lower LD₅.

9. EXAMPLE: DELETIONS AND INSERTIONS OF THE STALK OF THE INFLUENZA VIRUSNEURAMINIDASE: GENERATION OF A HOST RANGE MUTANT

Applying a reverse genetic system, the stalk of the influenza A/WSN/33virus neuraminidase (NA) gene was altered by making deletions,insertions and mutations in this region of the gene. The data presentedin this example show that the length of the NA stalk can be variable.Interestingly, a deletion of 28 amino acids resulted in a host rangemutant virus with a markedly reduced growth rate in MDCK cells ascompared to that in MDBK cells. Also, an insertion of 41 extra aminoacids into the stalk did not significantly interfere with viral growthin either MDCK or MDBK cells, suggesting that the stalk region cantolerate the introduction of long foreign epitopes.

9.1. MATERIALS AND METHODS 9.1.1. CELLS AND VIRUS

Madin-Darby bovine kidney (MDBK) cells were used for RNP transfection.Mardin-Darby canine kidney (MDCK) and MDBK cells were used forpreparation of viruses as well as for determination of viral growthcharacteristics. MDBK cells were grown in reinforced minimal essentialmedium (REM) (Whittaker, Walkersville, Md.) supplemented with 10% FCS(GIBCO, Gaithersburg, Md.), and MDCK cells were grown in minimalessential medium (MEM) (Whittaker) containing 10% FCS (GIBCO). MDBK andMDCK cells were maintained in REM and MEM containing 0.42% bovinealbumin (BA), respectively. Influenza WSN-HK (H1N2) virus, a reassortantvirus, was used for the RNP transfection experiment (Enami, et al.,1990, Proc. Natl. Acad. Sci. USA 87:3802-3805). This virus has the NAgene of influence A/Hong Kong/8/68 virus and other seven RNA segmentsfrom A/WSN/33 virus, and it does not grow in MDBK cells in the absenceof trypsin. WSN-HK virus was propagated in 11 day old embryonated hens'eggs and titrated in MDCK cells.

9.1.2. CONSTRUCTION OF NA MUTANTS

Clone pT3NAv which contains the cDNA of the influenza A/WSN/33 virus NAgene was described previously (Enami et al. 1990, Proc. Natl. Acad. Sci.USA 87:3802-3805). No unique restriction enzyme site was found in theregion encoding the stalk of the NA protein. In order to facilitate themanipulation of the stalk of the NA, plasmid pT3NAv had to be modified.One of the choices was destroy the Sty I/Dsa I site at nucleotide 875 sothat the Sty I site at nucleotide 169 and the Dsa I site at nucleotide253 became unique. For achieving this, the pT3NAv DNA was cut by Nco Iat nucleotide 875 and trimmed with mungbean nuclease using standardmethods (Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual,2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).The DNA was then digested by Pst I at nucleotide 926. The fragmentbetween nucleotides 875 and 926 was replaced by a polymerase chainreaction (PCR) fragment in which nucleotide 877 was changed by a silentmutation. The PCR fragment was made (Erlich, 1989, PCR Technology:Principles and Applications for DNA Amplification, Stockton Press) usingpT3NAv as template and oligonucleotides NA01 and NA02 as primers (TableV). After digestion with Pst I, the PCR fragment was ligated into thepT3NAv DNA which was digested by both Nco I and Pst I. The modifiedpT3NAv was called pT3NAmod. Using the pT3NAmod clone, a series of NAmutants with deletions, insertions and mutations in the stalk region ofthe NA protein were generated. The oligonucleotides used for makingthese constructs are shown in Table V:

TABLE V CONSTRUCTION OF NA MUTANTS Method of Mutants OligonucleotidesConstruction* T₃NAmod NA01 (SEQ ID NO:5) 5′-CCTGGGTGTCCTTC-3′ PCR NA02(SEQ ID NO:6) 5′-CCCCACTGCAGATG-3′ Del 16 NA03 (SEQ ID NO:7)5′-CAAGGCAGCACCGGCAA Annealing CTCGAGTCTTTGTCCATC-3′ NA04 (SEQ ID NO:8)5′-CACGGATGGGACAAAGA CTCGAGTTGCCGGTGCTGC-3′ Del 18m NA05 (SEQ ID NO:9)5′-GCGCGCTCGAGAGGCTGCC PCR TTGG-3′ M 13 (SEQ ID NO:10)5′-GTAAAACGACGGCCAGT-3′ Del 23N NA06 (SEQ ID NO:11)5′-GCGCGCTCGAGTTGCCGGTA PCR GTATGGTTTTG-3′ M 13 See Above Del 23C NAO7(SEQ ID NO:12) 5′-GCGCGCCACGGCAAAGAGATGA PCR ATTGTTGCATATTCC-3 M13 SeeAbove Del 28N NAO8 (SEQ ID NO:13) 5′-GCGCGCTCGAGTTGCCGGTTC PCRCGGTTTGAAT-3 M13 See Above N72Q/ NA09 (SEQ ID NO:14)5′-GCGCGCCACGGATGGGCGAAAGA PCR C76S GATGATTGGCCGGTTAATATC-3′ M13 SeeAbove Ins 12 NA10 (SEQ ID NO:15) 5′-CTTGGATGGGACAAAGACTCGAG AnnealingTTGCCGGTGCTGC-3′ NAO3 See Above Ins 41 NA11 (SEQ ID NO:16)5′-GCGCGCCGTGGGACCGGAA PCR ATC-3′ NA12 (SEQ ID NO:17)5′-AGCCCACCCACGG-3′ *For all PCR reactions, pT3NAV was used as template.The underlined nucleotides indicate the position of a newly created Ava1 site.

Del 16 was created by replacing the fragment between the Sty I and Dsa Isites in pT3NAmod with the annealed oligonucleotides NA03 and NA04.Through silent mutations, a unique Ava I site was created in plasmid Del16 (underlined nucleotides in Table V). Mutant Del 18m, Del 23N and Del28N was made by PCR using pT3NAv as template and M13/Puc forwardsequencing primer (5′-GTAAAACGACGGCCAGT-3′SEQ ID NO: 10) and NA05, NA06and NA08 as primer, respectively. The PCR products were digested by EcoRI and Ava I and inserted into plasmid Del 16 DNA which was digested bythe same enzymes. Mutant Del 23C and N72Q/C76S were also made by PCR inthe same way except for using NA07 and NA09 as primer, respectively. ThePCR products were digested with Dsa I and Eco RI, and inserted intopT3NAmod digested with the same enzymes. Ins. 12 and Ins 24 wereobtained by inserting the annealed oligonucleotides NA03 and NA10 intothe Sty I site of pT3NAmod. Ins 24 contains a duplicate of the insertionpresent in Ins 12. Ins 41 was made by PCR using pT3NAv as template andoligonucleotides NA11 and NA12 as primers. The PCR fragment was digestedby Dsa I and inserted into the Dsa I site of pT3NAmod. In order to makeDel 28 C, plasmid pT3NAmod was cut by Dsa I, and filled-in with reversetranscriptase (BRL, Bethesda, Md.). The DNA was then digested by Sty I,trimmed with mungbean nuclease and blunt-end ligated.

9.1.3. RNP TRANSFECTION

The RNP transfection experiments were carried out by using the standardprotocol as reported before (Enami and Palese, 1991, J. Virol.65:2711-2713). Briefly, the plasmid DNAs were digested by restrictionenzyme Ksp6321 or Ear I. 1 μg linearized DNA was incubated in 10 μlviral nucleoprotein and polymerase proteins (approximately 1 μg)isolated from influence A/PR/8/34 virus and 100 U T3 RNA polymerase(Stratagene, La Jolla, Calif.) in a 50 μl volume under transcriptionconditions. The in vitro reconstituted RNP complex was then transfectedinto MDBK cells which were infected with WSN-HK reassortant virus at anmode of infectivity (m.o.i.)=1. At 20 hours post infection, thesupernatant was collected and used for plaque assay in MDBK cells inorder to select the transfectant virus.

9.1.4. VIRUS PREPARATION AND RNA EXTRACTION

Transfectant viruses were isolated and amplified from individual plaquesin MDBK cells. Then viruses were propagated in 175 cm² large flasks ofMDCK cells, and purified through 30-60% sucrose gradients. Virion RNAwas extracted from purified viruses as described previously (Luo et al.,1992, J. Virol. 66:4679-4685).

9.1.5. ELECTROPHORESIS AND SILVER STAINING OF vRNA SEGMENTS

The vRNA extracted from viruses was analyzed on a 2.8% acrylamide gelcontaining 7.7 M urea. The vRNA segments were then visualized by silverstaining as reported before (Enami et al., 1990, Proc. Natl. Acad. Sci.USA 87:3802-3805).

9.1.6. REVERSE TRANSCRIPTION AND PCR

The NA-specific RNA of the viruses containing wild type NA or Del 18m,Del 23N and Del 28N mutant NAs were transcribed by reverse transcriptase(BRL, Bethesda, Md.) using oligonucleotide5′-GTCAATCTGTATGGTAGTCGG-3′(SEQ ID NO: 18) as primer, which coversnucleotide 52 to nucleotide 72. The reverse transcripts were amplifiedby PCR using the above oligonucleotide and oligonucleotide NA12 (TableV) as primers (Luo et al., 1991, J. Virol. 65:2861-2867). Primer NA12was labelled with gamma ³²[P]ATP. The PCR products were denatured byalkali and analyzed on a 6% polyacrylamide gel containing 7 M urea.

9.1.7. GROWTH CURVE

MDBK and MDCK cells were infected with wild type virus and each of thetransfectant viruses at an m.o.i.=0.001, and maintained in REM and MEMmedia containing 0.5 μg/ml trypsin. Supernatants were collected at 12,24, 36 and 48 hour postinfection. The number of plaque forming units(PFU) of each supernatant was determined by plaque assay in MDBK cells.

9.2. RESULTS

The influenza virus neuraminidase (NA) protein functions during theinfectious cycle as an enzyme to remove terminal sialic acids. Itsaction may prevent self-aggregation of virus particles, and promotevirus release during budding from host cells (Palese et al., 1974,Virol. 61:397-410). NA is an integral membrane glycoprotein anchored inthe viral membrane as a homo-tetramer. Each tetrameric NA appears as amushroom-shaped spike (FIG. 7) on the surface or the virion when viewedin the electron microscope (Laver and Valentine, 1969, Virol.38:105-119; Wrigley et al., 1973, Virol. 51:525-529). Structurally, themonomer of the NA consists of four different domains (FIG. 7): acytoplasmic and a transmembrane domain, the thin stalk, and the globularhead (Blok and Air, 1982, Biochem. 21:4001-4007; Colman, 1989, in “TheInfluenza Viruses”, R. M. Krug, ed., pp. 175-218, Plenum Press, NewYork). Although much is known about the structure and role of the headregion, the structure-function relationship of the stalk region is lesswell understood. Presented below is a study of the stalk region of theNA in which the length and structure of the stalk is varied bydeletions, insertions and mutations.

9.2.1. RESCUE OF DELETION MUTANTS OF NA INTO INFECTIOUS VIRUS

Based on sequence comparisons of different influenza A virus NA genes,it has been suggested that the stalk region of the NA varies between 25and 57 amino acids (Blok and Air, 1982, Biochem. 21:4001-4007; Blok andAir, 1982, Virol. 118:229-234; Els et al., 1985, Virol. 142:241-247;Colman, 1989, in “The Influenza Viruses”, R. M. Krug, ed., pp. 175-218,Plenum Press, New York ). The stalk region of the influenza A/WSN/33virus NA, which was used in this study, is approximately 41 amino acidslong (Blok and Air, 1982, Biochem. 21:4001-4007; Blok and Air, 1982,Virol. 118:229-234; Hiti and Hagar, 1982, J. Virol. 41:730-734; Colman,1989, in “The Influenza Viruses”, R. M. Krug, ed., pp. 175-218, PlenumPress, New York). It was first asked whether NA mutants with largedeletions could be rescued into infectious virus particles. The firstmutant gene, Del 16, (SEQ ID NO: 27) that was constructed lacked 48nucleotides (amino acid position 53-69) (FIG. 8), and following RNPtransfection infectious virus was isolated. Purified RNA was separatedon a 2.8% acrylamide gel containing 7.7 M urea and the NA segment withthe 48 nucleotide deletion (Del 16) was shown to migrate faster than theNA RNA of the wild type virus (FIG. 9: compare lane 1 and lane 2). Thissuggested that influenza A/WSN/33 virus can tolerate a neuraminidasewith a stalk of only about 25 amino acids. A gene encoding an NA with a28 amino acid deletion (Del 28C, (SEQ ID NO: 28) FIG. 8) was thenconstructed. After RNP transfection, however, no infectious virus wasrescued. This result could be either due to the truncation of the stalkregion or due to the deletion of a potential glycosylation site atposition 74 and/or of a cysteine at position 76. Since the potentialglycosylation site and the cysteine residue are highly conserved amongdifferent NAs (Blok and Air, 1982, Biochem. 21:4001-4007), thepossibility was first explored that important structural elements weredestroyed by this deletion. For this purpose, three other NA mutantswere constructed: N72Q/C76S, Del 23C, and Del 18m (SEQ ID NO: 29) (FIG.8). In the N72Q/C76S mutant, the asparagine (N) at position 72 and thecysteine (C) at position 76 were mutated into glutamine (Q) and serine(S), respectively. Although this gene has the same length of the stalkas the wild type gene, no infectious virus could be rescued. To dissectthe contribution of the glycosylation site and of the cysteine residue,mutant Del 18m was constructed by changing the asparagine at position 72to leucine and deleting amino acids 53 to 71. Interestingly, infectiousprogeny virus was obtained following RNP transfection of the Del 18m NARNA. This finding suggests that the cysteine at position 76, rather thanthe potential glycosylation site at position 72 is essential for theformation of infectious virus. However, it was not clear whether or notdeletions beyond the cysteine can be tolerated. Thus mutant Del 23C (SEQID NO: 30) was designed in which the potential glycosylation site andthe cysteine were reintroduced into the NA but two amino acidsC-terminal to the cysteine were deleted. No virus containing the Del 23Cmutant gene was obtained. It thus appears that the sequence followingthe stalk region does not tolerate deletions, suggesting that thisregion is part of the NA “head”. However, the possibility could not beruled out that the Del 23C mutant virus was non-infectious due to thefurther shortening of the NA stalk by 5 amino acids. Two more constructswere therefore created: Del 23N (SEQ ID NO: 31) and Del 28N (SEQ ID NO:32). The Del 23N mutant has a 23 amino acid deletion between amino acid47 and 69, and the Del 28N mutant has a 28 amino acid deletion startingat amino acid 42 and extending to amino acid 69. Both Del 23N and Del28N resulted in infectious influenza viruses. This suggests that an NAstalk of approximately 12 amino acids suffices to generate infectiousvirus. The deletion analysis was not extended further because the viruscontaining the Del 28N mutant NA does not grow as well as wild typevirus (see below), and has a deletion that leaves only a few amino acidsnear the viral membrane at the N-terminal and the cysteine at theC-terminal side. In order to verify that the NA RNAs of the mutantviruses had truncated stalk regions, the NA specific RNAs were reversetranscribed and analyzed by PCR using a labelled primer. The sizes ofthe PCR products of wild type, Del 18m, Del 23N and Del 28N were asexpected (FIG. 10, lanes 2-5).

9.2.2. RESCUE OF INSERTION MUTANTS OF NA INTO INFECTIOUS VIRUS

The deletion analysis revealed that the length of the NA stalk isflexible. Virus is still viable even when it contains an NA whose stalkregion is almost completely deleted. The question, however, remained asto whether or not the stalk of the NA molecule can tolerate theinsertion of extra amino acids. To answer this 12 and 24 amino acidswere inserted into the stalk region of the NA between amino acids 50 and51, as shown in FIG. 8 (Ins. 12 (SEQ ID NO: 33) and Ins. 24 (SEQ ID NO:34), respectively). After RNP transfection, both Ins 12 and Ins 24mutant NA RNAs were rescued into infectious transfectant viruses. Thefact that these two NA mutants were viable, led the insertion ofadditional amino acids. To that end, mutant Ins 41 NA was generated byinserting 41 amino acids between position 39 and 40. The Ins 41 (SEQ IDNO: 35) mutant contains a duplication in the NA stalk. A gain,infectious virus was rescued, indicating that the NA stalk tolerates a41 amino acid insertion. The purified RNAs of Ins 12, Ins 24 and Ins 41mutant viruses were analyzed on a polyacrylamide gel and the rescued NARNAs were found to migrate in the expected positions (FIG. 9, lanes 3,4, and 5).

9.2.3. GROWTH CHARACTERISTICS OF THE NA TRANSFECTANT VIRUSES

In order to determine whether deletions and insertions in the stalkregion of the NA molecule have effects on virus growth, the transfectantviruses were characterized in MDBK and MDCK cells. The results are shownin FIGS. 11A-11B. Since all mutant viruses were rescued by isolatingprogeny viruses in MDBK cells to allow selection against the helpervirus, it is not surprising that all mutants grow in this cell type(FIG. 11A). It appears that mutant Del 28N and mutant Ins 24 growslightly slower and to lower titers than do either the remaining mutantsor the wild type virus. It should also be noted that the MDBK cell linecurrently in use yields lower overall titers than other MDBK cell lines.The yields in MDCK cells (FIG. 11B) are higher by approximately one log,except those of mutant Del 18m and mutant Del 28N, which grow to 10² and10⁴ time lower titers, respectively, than does wild type virus. MutantDel 28N is thus a host range mutant which grows about 1,000 times lessefficiently in MDCK cells as compared to MDBK cells.

The present invention is not to be limited in scope by the specificembodiments described which are intended as single illustrations ofindividual aspects of the invention, and any constructs or viruses whichare functionally equivalent are within the scope of this invention.Indeed, various modifications of the invention in addition to thoseshown and described herein will become apparent to those skilled in theart from the foregoing description and accompanying drawings. Suchmodifications are intended to fall within the scope of the appendedclaims.

36 12 amino acids amino acid unknown peptide not provided 1 Asn Glu AspSer Tyr Val Pro Ser Ala Glu Gln Ile 1 5 10 21 base pairs nucleic acidsingle linear DNA not provided 2 GGGAACAATT AGGTCAGAAG T 21 21 basepairs nucleic acid single linear DNA not provided 3 GTGGCAATAACTAATCGGTC A 21 10 amino acids amino acid unknown peptide not provided 4Pro Ala Ser Thr Thr Asn Lys Asp Lys Leu 1 5 10 14 base pairs nucleicacid single linear DNA not provided 5 CCTGGGTGTC CTTC 14 14 base pairsnucleic acid single linear DNA not provided 6 CCCCACTGCA GATG 14 36 basepairs nucleic acid single linear DNA not provided 7 CAAGGCAGCACCGGCAACTC GAGTCTTTGT CCCATC 36 36 base pairs nucleic acid single linearDNA not provided 8 CACGGATGGG ACAAAGACTC GAGTTGCCGG TGCTGC 36 23 basepairs nucleic acid single linear DNA not provided 9 GCGCGCTCGAGAGGCTGCCT TGG 23 17 base pairs nucleic acid single linear DNA notprovided 10 GTAAAACGAC GGCCAGT 17 31 base pairs nucleic acid singlelinear DNA not provided 11 GCGCGCTCGA GTTGCCGGTA GTATGGTTTT G 31 37 basepairs nucleic acid single linear DNA not provided 12 GCGCGCCACGGCAAAGAGAT GAATTGTTGC ATATTCC 37 31 base pairs nucleic acid singlelinear DNA not provided 13 GCGCGCTCGA GTTGCCGGTT CCGGTTTGAA T 31 44 basepairs nucleic acid single linear DNA not provided 14 GCGCGCCACGGATGGGCGAA AGAGATGATT GGCCGGTTAA TATC 44 36 base pairs nucleic acidsingle linear DNA not provided 15 CTTGGATGGG ACAAAGACTC GAGTTGCCGGTGCTGC 36 22 base pairs nucleic acid single linear DNA not provided 16GCGCGCCGTG GGACCGGAAA TC 22 13 base pairs nucleic acid single linear DNAnot provided 17 AGCCCACCCA CGG 13 21 base pairs nucleic acid singlelinear DNA not provided 18 GTCAATCTGT ATGGTAGTCG G 21 54 base pairsnucleic acid single unknown RNA not provided 19 UUACAAACAA GUUUUUUGAGGAACAAAGAU GAUCGUUUGC GUCCUCAAAC UUAC 54 53 base pairs nucleic acidsingle unknown RNA not provided 20 AGUAGAAACA AGGAGUUUUU UGAACAAACUACAUUUAAAC UCCUGCUUUC GCU 53 78 base pairs nucleic acid single unknownRNA not provided 21 AGUAGUAACA AGAGGAUUUU UAUUUUACAU UCACAUCUUUCCGUUUGCCA GUGACUAAAU 60 AAAUCCUCUG CUUCUGCU 78 13 amino acids aminoacid unknown peptide not provided 22 Cys Asp Ser Leu Leu Pro Ala Arg SerTrp Ser Tyr Ile 1 5 10 17 amino acids amino acid unknown peptide notprovided 23 Cys Asn Glu Asp Ser Tyr Val Pro Ser Ala Glu Gln Ile Trp SerTyr 1 5 10 15 Ile 13 amino acids amino acid unknown peptide not provided24 Trp Leu Thr Lys Lys Gly Asp Ser Tyr Pro Lys Leu Thr 1 5 10 23 aminoacids amino acid unknown peptide not provided 25 Trp Leu Thr Lys Lys GlyPro Ala Ser Thr Thr Asn Lys Asp Lys Leu 1 5 10 15 Asp Ser Tyr Pro LysLeu Thr 20 45 amino acids amino acid unknown peptide not provided 26 HisSer Ile Gln Thr Gly Asn Gln Asn His Thr Gly Ile Cys Asn Gln 1 5 10 15Gly Ser Ile Thr Tyr Lys Val Val Ala Gly Gln Asp Ser Thr Ser Val 20 25 30Ile Leu Thr Gly Asn Ser Ser Leu Cys Pro Ile Arg Gly 35 40 45 29 aminoacids amino acid unknown peptide not provided 27 His Ser Ile Gln Thr GlyAsn Gln Asn His Thr Gly Ile Cys Asn Gln 1 5 10 15 Gly Ser Thr Gly AsnSer Ser Leu Cys Pro Ile Arg Gly 20 25 17 amino acids amino acid unknownpeptide not provided 28 His Ser Ile Gln Thr Gly Asn Gln Asn His Thr GlyIle Cys Asn Arg 1 5 10 15 Gly 27 amino acids amino acid unknown peptidenot provided 29 His Ser Ile Gln Thr Gly Asn Gln Asn His Thr Gly Ile CysAsn Gln 1 5 10 15 Gly Ser Leu Ser Ser Leu Cys Pro Ile Arg Gly 20 25 22amino acids amino acid unknown peptide not provided 30 His Ser Ile GlnThr Gly Asn Gln Asn His Thr Gly Ile Cys Asn Asn 1 5 10 15 Ser Ser LeuCys Arg Gly 20 22 amino acids amino acid unknown peptide not provided 31His Ser Ile Gln Thr Gly Asn Gln Asn His Thr Thr Gly Asn Ser Ser 1 5 1015 Leu Cys Pro Ile Arg Gly 20 17 amino acids amino acid unknown peptidenot provided 32 His Ser Ile Gln Thr Gly Thr Gly Asn Ser Ser Leu Cys ProIle Arg 1 5 10 15 Gly 19 amino acids amino acid unknown peptide notprovided 33 Cys Asn Gln Gly Ser Thr Gly Asn Ser Ser Leu Cys Pro Ile GlnGly 1 5 10 15 Ser Ile Thr 31 amino acids amino acid unknown peptide notprovided 34 Cys Asn Gln Gly Ser Thr Gly Asn Ser Ser Leu Cys Pro Ile GlnGly 1 5 10 15 Ser Thr Gly Asn Ser Ser Leu Cys Pro Ile Gln Gly Ser IleThr 20 25 30 43 amino acids amino acid unknown peptide not provided 35Gln Thr Gly Asn Gln Asn His Thr Gly Ile Cys Asn Gln Gly Ser Ile 1 5 1015 Thr Tyr Lys Val Val Ala Gly Gln Asp Ser Thr Ser Val Ile Leu Thr 20 2530 Gly Asn Ser Ser Leu Cys Pro Ile Arg Gly Thr 35 40 44 amino acidsamino acid unknown peptide not provided 36 His Ser Ile Gln Thr Gly AsnGly Asn His Thr Gly Ile Cys Asn Gln 1 5 10 15 Gly Ser Ile Thr Val LysTrp Ala Gly Gln Asp Ser Thr Ser Val Ile 20 25 30 Leu Thr Gly Gln Ser SerLeu Ser Pro Ile Arg Gly 35 40

What is claimed is:
 1. An attenuated genetically engineereddouble-stranded segmented RNA virus containing at least one modificationin the conserved sequences of the 5′ or 3′ termini of a viral genesegment that down-regulates transcription of at least one viral geneencoded by the gene modified segment, and results in the production offewer progeny virions than would be generated by a corresponding wildtype virus.
 2. The attenuated virus of claim 1 which belongs to thereovirus family.
 3. The attenuated virus of claim 2 in which a viralnon-structural gene is modified.
 4. The attenuated virus of claim 1 inwhich the modified termini down-regulates transcription of the viralenvelope gene.
 5. The attenuated virus of claim 1 in which the modifiedtermini down-regulates transcription of the viral protease gene.
 6. Theattenuated virus of claim 1 in which the modified termini down-regulatestranscription of the viral polymerase gene.